NGS Sequencing

Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.

Targeted Testing

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Glutaric acidemia II patients are split between those with causative variants in the ETFA, ETFB and ETFDH genes, and it is difficult to estimate the fraction with variants in each gene. However, to date all reported causative variants in the ETFA gene are detectable via direct sequencing, and in studies with somewhat larger numbers of patients with ETFA variants the majority had two variants identified (for example, 23 alleles found in 13 patients, for an overall analytical sensitivity of ~88%; Schiff et al. 2006).

Deletion/Duplication Testing via aCGH

Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

To date, no large deletions or duplications have been described in the ETFA gene, although most studies of glutaric acidemia type II patients that included ETFA analysis have not been reported to have included deletion and duplication testing.

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Clinical Features

Glutaric acidemia (GA) type II, also known as multiple acyl-CoA dehydrogenase deficiency (MADD), is an inherited disorder of fatty acid and amino acid oxidation. GA type II is caused by defects in one of three genes (ETFA, ETFB or ETFDH). Clinical and biochemical features are not typically useful in distinguishing which gene is affected in GA type II patients. Three sub-categories of GA type II are recognized, each of which is based on severity and presentation of clinical symptoms (Frerman and Goodman 2014).

Patients in the first group present within the first 48 hours of life with severe hypoketotic hypoglycemia, hypotonia, metabolic acidosis, possibly hepatomegaly and a “sweaty feet” odor similar to that observed in isovaleric acidemia patients, and multiple congenital anomalies, such as dysplastic kidneys, facial dysmorphism, rocker bottom feet, abdominal wall defects and abnormal external genitalia. Such patients typically die within the first week of life (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).

Patients in the second group present similarly to those in the first group, but without congenital anomalies. If these patients survive beyond the first week of life, they typically succumb within the first few months, often due to cardiomyopathy (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).

The patients in the third group have a more mild form of GA type II, with onset anywhere from infancy to adulthood. Clinical presentation also varies widely in this group, and may include vomiting, hypoglycemia, metabolic acidosis, hepatomegaly, progressive lipid storage proximal myopathy and exercise intolerance. Symptoms in the third group often present in an episodic manner, making biochemical analysis challenging as abnormalities may be detectable only during periods of metabolic crisis (Olsen et al. 2003; Schiff et al. 2006; Frerman and Goodman 2014; Xi et al. 2014).

In all GA type II patients, generalized aminoacidemia and aminoaciduria, hyperammonemia and metabolic acidosis may be observed. Increased levels of sarcosine in both the serum and urine are common in those with mild, later onset GA type II. Biochemically, GA type II can be distinguished from GA type I based on the presence of 2-hydroxyglutaric acid (3-hydroxyglutaric acid is present in GA type I patients) (Frerman and Goodman 2014).

To date, no effective treatments are available for patients with the severe, infantile onset forms of GA type II. Some patients with the mild, later onset form have been shown to respond well to riboflavin, glycine and L-carnitine supplementation, as well as to dietary restriction of fat and protein (Olsen et al. 2007; Wen et al. 2013; Frerman and Goodman 2014). The majority of patients reported with the late-onset, riboflavin responsive form of GA type II have had defects in the ETFDH gene (Olsen et al. 2007; Wen et al. 2010; Cornelius et al. 2012; Wen et al. 2013).

Genetics

Glutaric acidemia type II is an autosomal recessive disorder caused by pathogenic variants in the ETFA, ETFB or ETFDH genes. To date, over 25 causative variants have been reported in the ETFA gene (Human Gene Mutation Database). The majority of reported pathogenic variants are missense, although nonsense variants, small deletions, insertions and duplications have all been reported. The variants are spread throughout the gene, and the most commonly reported variant is Thr266Met (Schiff et al. 2006; Frerman and Goodman 2014).

The ETFA gene is located on chromosome 15 and contains 12 exons. ETFA encodes the a-subunit of the electron transfer flavoprotein (ETF). The ETF protein is found in the mitochondrial matrix as a heterodimer, comprised of one subunit each of α- and β-monomers. ETF accepts electrons from at least 12 other flavoprotein dehydrogenases, then transfers them to the mitochondrial respiratory chain via the electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), encoded by the ETFDH gene. Clinical symptoms observed in ETFA deficient patients are due to a disruption of electron transfer to the respiratory chain (Cornelius et al. 2012; Frerman and Goodman 2014).

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

This test provides full coverage of all coding exons of the ETFA gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

Individuals with a positive newborn screening result for glutaric acidemia type II are good candidates for this test, as are individuals that exhibit biochemical and clinical symptoms of GA type II. Family members of patients known to have ETFA variants are also good candidates, and we will also sequence the ETFA gene to determine carrier status.

TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Ship blood tubes at room temperature in an insulated container. Do not freeze blood.

During hot weather, include a frozen ice pack in the shipping container.
Place a paper towel or other thin material between the ice pack and the blood tube.

In cold weather, include an unfrozen ice pack in the shipping container as insulation.

At room temperature, blood specimen is stable for up to 48 hours.

If refrigerated, blood specimen is stable for up to one week.

Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.

For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.

DNA may be shipped at room temperature.

Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.

We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

PreventionGenetics should be notified in advance of arrival of a cell culture.

Culture and send at least two T25 flasks of confluent cells.

Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.

Send specimens in insulated, shatterproof container overnight.

Cell cultures may be shipped at room temperature or refrigerated.

Label the flasks with the patient name, date of birth, and/or ID number.

We strongly recommend maintaining a local back-up culture. We do not culture cells.